Transcription of protein-encoding genes in eukaryotes is a highly regulated process that is initiated when a myriad of transcriptional activators recognize specific DNA elements proximal and distal to core promoters. These DNA bound activators then direct the dynamic assembly of RNA polymerase II, general transcription factors, and cofactor complexes on promoter DNA to initiate transcription. As the exact sequence of events needed to initiate transcription is poorly understood, a mechanistic understanding of how transcription activators regulate and direct transcription complex formation from their proximal and distant recognition sites is crucial to understanding how genes expression is regulated. Toward a better understanding of the mechanism of RNAPII mediated transcription we will develop and apply single molecule techniques to (i) measure the rates and the sequence by which the GTFs and RNAPII bind to promoter DNA, (ii) determine how the rates and the sequence by which the GTFs and RNAPII bind to promoter DNA are modulated by transcriptional activators and co- activators, and (iii) determine how activation occurs by distal enhancers. Information on dynamics of transcription initiation obtained in these single-molecule studies will complement the structural information obtained by the proposed biochemical, EM, and x-ray crystallographic studies of this PPG. Mechanistic understanding of transcription complex formation will provide new potential protein targets for anticancer therapies.